Not applicable.
Within the United States, ongoing research is directed toward developing alternative energy sources to reduce our dependence on foreign oil and nonrenewable energy. The use of ethanol as a fuel has become increasingly prevalent in recent years. The current domestic use of ethanol in transportation fuels is about 1.2 billion gallons annually. In the U.S., the majority of ethanol is obtained from the fermentation of cornstarch. Projections made by the Department of Energy indicate that by the year 2020, annual ethanol usage in fuels will have increased dramatically to an estimated 20 billion gallons. This greatly exceeds what can be economically produced from cornstarch.
In order to meet the increased demand for ethanol, it will be necessary to ferment sugars from other biomass. Biomass refers to materials such as agricultural wastes, corn hulls, corncobs, cellulosic materials and the like. Biomass from most of these sources contains xylose at a concentration of up to about 25-30% of the total dry weight. The D-xylose content of hardwood species and herbaceous angiosperms is about 17% and 31% of the total dry weight, respectively. Because agricultural residues, pulping wastes, and fast-growing hardwood species have a high xylose content, the potential economic and ecologic benefits of converting xylose in these renewable materials are significant. In order for biomass conversion to be economically feasible, a practical, large-scale use must be found for xylose.
Biomass conversion employs microorganisms that serve as biocatalysts to convert cellulosic materials into usable end products such as ethanol. Efficient biomass conversion in large-scale industrial applications requires a microorganism that can tolerate high sugar and ethanol concentrations, and which is able to ferment multiple sugars simultaneously.
The pentoses D-xylose and L-arabinose are among the most difficult sugars in biomass to metabolize. Bacteria can ferment pentoses to ethanol and other co-products, and bacteria with improved ethanol production from pentose sugars have been genetically engineered. However, these bacteria are sensitive to low pH and high concentrations of ethanol, their use in fermentations is associated with co-product formation, and the level of ethanol produced remains too low to make the use of these bacteria in large-scale ethanol production be economically feasible.
In general, industrial producers of ethanol strongly favor the use of yeast as biocatalysts, because yeast fermentations are relatively resistant to contamination, are relatively insensitive to low pH and ethanol, and are easier to handle in large-scale processing. Many different yeast species use xylose respiratively, but only a few species use xylose fermentatively. Fermentation of xylose to ethanol by wild type xylose-fermenting yeast species occurs slowly and results in low yields, relative to fermentation rates and ethanol yields that are obtained with conventional yeasts in glucose fermentations. In order to improve the cost effectiveness of xylose fermentation, it is necessary to increase the rate of fermentation and the ethanol yields obtained.
The most commonly used yeast in industrial applications is Saccharomyces cerevisiae. Although S. cerevisiae is unable to grow on or ferment xylose, it was reported that homogenates of S. cerevisiae could readily ferment D-ribulose-5-phosphate to ethanol, and that it could also convert D-xylulose-5-phosphate to a lesser extent (Dickens, 1938). Efforts to create strains of S. cerevisiae with enhanced xylose fermentation by introducing genes capable of converting xylose to metabolites fermentable by S. cerevisiae have been largely unsuccessful (Ueng et al., 1985; Chan et al., 1989; Amore et al., 1989; Sarthy et al., 1987; Toivari et al., 1996).
Pichia stipitis is a yeast species that is able to ferment xylose to produce ethanol. In P. stipitis, fermentative and respirative metabolism co-exist to support cell growth and the conversion of sugar to ethanol (Ligthelm, 1988). P. stipitis differs significantly from the glucose-fermenting yeast S. cerevisiae in its ability to produce ethanol from xylose. It is known that P. stipitis requires a well-controlled low level of oxygen to reach maximum rate of ethanol production. In 1996, Passoth et al. first observed a peculiar pattern of respiration in P. stipitis. After the cells of P. stipitis were transferred from aerobic to oxygen-limited conditions, no decrease in the respiration capacity was observed. In addition, there was no increase in the respirative quotient (CO2 production/O2 consumption), and no change in the level of a key respiratory enzyme, pyruvate dehydrogenase. Moreover, respiratory activity was not repressed in the presence of fermentable sugars or low oxygen tension. In a survey of alternative pathways present in Crabtree-positive and -negative species, Jeppsson et al. (1995) reported that P. stipitis has an alternative respiratory pathway that is resistant to cyanide or antimycin A, but is sensitive to salicyl hydroxymate (SHAM). The pathway is believed to include a SHAM-sensitive terminal oxidase (STO). Jeppsson et al. hypothesized that the STO pathway would serve as a redox sink to avoid the accumulation of xylitol in P. stipitis. 
Although STO respiration was discovered 70 years ago (Keilin, 1929), the physiological roles and the functional components of this pathway remain unclear. STO respiration has been widely reported from higher plants (for review, Douce and Neuburger, 1989; Vanlerberghe and McIntosh, 1997), fungi (Lambowitz and Slayman, 1971; Downie and Garland, 1973), and yeasts (Lloyd and Edwards, 1977). The STO pathway branches from the conventional cytochrome pathway at the level of ubiquinone, just before cytochrome b (Seidow, 1980; Storey, 1976), where electrons are directly donated to Sto to reduce molecular oxygen to water. Sto is unable to translocate protons (Moore and Rich, 1985), thus it by-passes two out of the three energy-generating sites in plants. Therefore, it is considered as an energy-wasting pathway. Most of the current information concerning biochemical and regulatory aspects of the pathway has been obtained from the studies in plant Sto proteins. Because this protein is tightly associated with the mitochondrial inner membrane, no pure forms have been obtained as yet for characterization studies of the metal center and kinetics. Moreover, the plant Sto proteins lose activity when they are solublized. These difficulties have hindered progress in understanding the physiological roles of the STO pathway.
Research involving the identification and characterization of STO protein and its role in P. stipitis was undertaken in our laboratory. The information that resulted from these efforts has allowed us to develop genetically engineered, xylose-fermenting yeast strains with enhanced ethanol production from xylose.
One aspect of the present invention is an isolated polynucleotide comprising a sequence encoding the Pichia stipitis SHAM-sensitive terminal oxidase of SEQ ID NO:26. Preferably, the isolated polynucleotide of the present invention is an isolated DNA molecule comprising the sequence of SEQ ID NO:25.
In another aspect, the present invention includes a vector comprising a polynucleotide comprising a sequence encoding Pichia stipitis SHAM-sensitive terminal oxidase, SEQ ID NO:26.
The present invention provides a genetic construct comprising a sequence encoding the Pichia stipitis SHAM-sensitive terminal oxidase of SEQ ID NO:26 operably connected to a promoter functional in yeast.
The present invention provides a xylose-fermenting mutant of a yeast or fungal species, the mutant having reduced SHAM-sensitive terminal oxidase relative to the levels of SHAM-sensitive terminal oxidase in the parent strain from which the mutant was derived, wherein the species is selected from the group consisting of Pichia stipitis, Group I species and Group II species, wherein Group I species natively comprise a cytochrome pathway and a SHAM-sensitive pathway, and wherein Group II species natively comprise a cytochrome pathway, an antimycin A- and SHAM-insensitive pathway, and a SHAM-sensitive pathway. Preferably, the mutant yeast strain is a P. stipitis mutant.
In one embodiment, the mutant xylose-fermenting yeast is a P. stipitis SHAM-sensitive terminal oxidase disruptant. Preferably, the P. stipitis SHAM-sensitive terminal oxidase disruptant is derived from FPL-UC7 (NRRL 21448). More preferably still, the P. stipitis SHAM-sensitive terminal oxidase disruptant is FPL-Shi 31 (NRRL Y-30230).
In an alternative embodiment, the mutant xylose-fermenting yeast comprises a construct expressing SHAM-sensitive terminal oxidase antisense mRNA.
A further aspect of the invention is a method for producing ethanol from the fermentation of xylose comprising the steps of culturing a mutant of xylose-fermenting yeast in a xylose-containing medium under suitable conditions for a period of time sufficient to allow fermentation of xylose to ethanol, the mutant yeast having reduced SHAM-sensitive terminal oxidase relative to the wild type parental strain from which the mutant is derived.
The present invention includes a recombinant yeast comprising a sequence encoding the Pichia stipitis SHAM-sensitive terminal oxidase of SEQ ID NO:26, the coding sequence operably connected to a promoter functional in yeast, the yeast belonging to a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces byanus, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces sake, Saccharomyces thermotolerans, Saccharomyces urvarum and Group III species, wherein the recombinant yeast expresses SHAM-sensitive terminal oxidase and wherein expression of the SHAM-sensitive terminal oxidase is correlated with cyanide-resistant respiration, the yeast species natively comprising an antimycin A-insensitive pathway and a cytochrome pathway.
It is an advantage that the mutant xylose-fermenting yeast of the present invention ferments xylose at an increased rate, relative to the parent strain having wild-type levels of SHAM-sensitive terminal oxidase.
Other advantages and features of the present invention will become apparent upon review of the specification.